Gastric acid secretion from parietal cells is essential for food digestion and pathogen elimination in the stomach. Dysregulation of gastric acid homeostasis underlies a spectrum of acid-related diseases, including atrophic gastritis, gastric cancer, peptic and duodenal ulcers, and gastroesophageal reflux disease (GERD); GERD alone affects at least 20% of the US population. Histamine, the primary transmitter that ?switches on? H+ secretion into the stomach lumen, acts by relocating the H+-K+-ATPase proton pump from cytoplasmic tubulovesicles (TVs) onto the apical canalicular membranes via vesicular transport and fusion. However, the mechanisms by which histamine induces the exocytosis of TVs remain unclear. Most types of regulated exocytosis, including neurotransmitter release, are Ca2+-dependent, but it remains controversial whether Ca2+ is involved in histamine-triggered TV exocytosis. Human mutations of Transient Receptor Potential Mucolipin-1 (ML1), a Ca2+-permeable channel of intracellular membranes, cause achlorhydria (low acid secretion). Using super-resolution confocal imaging, and by developing a new patch-clamp method to record directly from canalicular and tubulovesicular membranes and a new organelle-targeted Ca2+ imaging method to detect Ca2+ release from TVs, we identified ML1 as a likely histamine-triggered Ca2+ release channel in TVs. The central goal of this proposal is to investigate the roles of ML1 in TV exocytosis and to use mouse models to explore the potential clinical use of ML1 agonists and inhibitors in controlling acid secretion in vivo. Using an integrative approach with Ca2+ imaging, electrophysiology, voltage imaging, electron microscopy, small molecule channel agonists and inhibitors, and transgenic mouse models, we will test the hypothesis that histamine-cAMP-PKA signaling activate ML1 and the TV K+ channel KCNQ1 to trigger TV exocytosis and acid secretion. Aim 1 is to test the roles of ML1 and PKA in histamine-induced secretion of gastric acids. Aim 2 is to investigate the roles of KCNQ1 channels in histamine-induced gastric acid secretion. Finally, Aim 3 is to determine the roles of ML1 and KCNQ1 channels in controlling gastric acid levels in vivo. Our long-term goal of the proposed research is to lay the groundwork necessary to develop new therapeutic strategies for acid-related diseases.